RNA interference, which suppresses gene expression by small interfering RNA (siRNA), has been utilized in the medical field, but specific delivery of siRNA to cells or tissues remains challenging. Now, main method of siRNA... [ view full abstract ]
RNA interference, which suppresses gene expression by small interfering RNA (siRNA), has been utilized in the medical field, but specific delivery of siRNA to cells or tissues remains challenging. Now, main method of siRNA delivery is the method of using liposome. But liposomes can’t reach deep in the tumor tissue because the size of liposomes is several ten nm to several hundred nm. In contrast, recombinant small antibodies, such as single-chain fragment of variable region (scFv) and diabody consisting of the variable regions of antibody which has high specificity to targeted cells and tissues, can more highly permeate tissues and reach various disease sites. In this study, we aim to design the small antibodies bearing the siRNA to construct a drug delivery system (DDS) of siRNA (Fig. 1).
First, we prepared 20 recombinant small antibodies (10 scFvs and 10 diabodies) with cationic peptide fragments as RNA carrier at C-terminus via 2 types of linkers by means of E.coli expression system. The charged peptides electrostatically interact with negatively-charged siRNAs. SDS-PAGE and western blot analyses revealed low expression of the antibodies with LK15 peptide and protamine in E.coli, whereas the antibodies with Arg9, TAT and truncated protamine (tp) peptides were expressed and secreted in culture supernatant. The expressed scFv with tp was purified by immobilized metal affinity chromatography followed by size exclusion and cation exchange chromatographies; as a result, pure scFv with tp was obtained, but the yield of purified scFv with tp was 4 µg/L-culture, which was significantly low. Next, we tried to prepare recombinant small antibodies with RNA carrier by chemical conjugation using amine coupling. Antibodies after chemical conjugation with RNA carrier were fractionated by cation exchange chromatography. As a result, many small antibodies remained un-conjugated and also caused multimerization (Fig. 2).
In conclusion, the fusion of RNA carrier to small antibody decreased the expressed yield in E.coli. We are in progress of preparing small antibody-RNA carrier complex by means of site-specific chemical conjugation using enzyme with small antibody and RNA carrier.
